Fourteen-Day and Thirteen-Week Studies:
In the 14-day studies (0, 310, 620, 1,250, 2,500, or 5,000 ppm), all five male and five female rats that received 5,000 ppm and 1/5 male rats that received 2,500 ppm died before the end of the studies; all mice lived to the end of the study. The final mean body weights of rats that received 2,500 ppm were lower than the initial weights. The final mean body weights of mice that received 2,500 or 5,000 ppm were 8% or 18% lower than that of controls for males and 2% or 8% lower for females.
In the 13-week studies, all rats lived to the end of the studies (dietary concentrations of 0 or 120-2,000 ppm). The final mean body weights of rats that received 500, 1,000 or 2,000 ppm were 12%, 13%, or 32% lower than that of controls for males and 4%, 8%, or 20% lower for females. Feed consumption by rats that received 2,000 ppm was somewhat lower than that by controls. Bone marrow atrophy was observed at increased incidences and severity in dosed rats. In the 13-week study (0 or 500-8,000 ppm), 1/10 male mice that received the highest concentration died before the end of the studies. The final mean body weights of mice that received 8,000 ppm were lower than the initial mean body weights. The final mean body weights of male mice that received 4,000 ppm and of female mice that received 2,000 or 4,000 ppm were 13%-19% lower than those of controls. Feed consumption was not related to dose. Minimal-to-moderate cytoplasmic vacuolization of hepatocytes was seen in 8/10 male mice that received 8,000 ppm.
Based on these results, dietary concentrations selected for the 2-year studies were 0, 120, or 250 ppm rhodamine 6G for rats, 0, 1,000, or 2,000 ppm for male mice, and 0, 500, 1,000 ppm for female mice.
Body Weight and Survival in the Two-Year Studies:
Mean body weights of dosed rats were similar to those of controls throughout the studies. The average daily feed consumption by dosed rats was within 5% that by controls for all dosed groups. The average amount of rhodamine 6G consumed per day was approximately 5 mg/kg for low dose rats and 10 or 12 mg/kg for high dose male or female rats. Mean body weights of high dose male and dosed female mice were generally 5%-14% lower than those of controls. The average daily feed consumption by dosed mice was within 5% that by controls for all dosed groups. The average amount of rhodamine 6G consumed per day was approximately 210 or 440 mg/kg for low dose or high dose male mice and 125 or 250 mg/kg for low dose or high dose female mice. No significant differences in survival were observed between any groups of rats or mice (male rats: control, 22/50; low dose, 21/50; high dose, 27/50; female rats: 29/50; 30/50; 30/50; male mice: 36/50; 32/50; 38/50; female mice: 39/50; 35/50; 36/50).
Nonneoplastic and Neoplastic Effects in the Two-Year Studies:
No chemically related nonneoplastic lesions in male or female rats and no chemically related neoplastic or nonneoplastic lesions in male or female mice were observed in these studies.
The incidences of keratoacanthomas of the skin was increased in high dose male rats (control, 1/50; low dose, 2/50; high dose, 8/50). The historical incidence of keratoacanthomas in untreated control male F344/N rats is 31/1,936 (1.6%; range, 0/50-7/49). Both fur and skin of rats in the dosed groups apparently were exposed to feed dust containing rhodamine 6G; the intensity of staining was proportional to the concentration of rhodamine 6G in feed. Because of the variable background incidence of keratoacanthomas in F344/N rats, the incidence of keratoacanthomas cannot be conclusively related to exposure to rhodamine 6G.
The incidences of pheochromocytomas (3/50; 3/50; 8/50) or malignant pheochromocytomas (combined: 3/50; 3/50; 10/50) of the adrenal gland were increased in high dose female rats. The historical incidence of adrenal medullary neoplasms in untreated control F344/N female rats is 99/1,968 (5%; range, 0/50-8/50). This marginal increase may be related to the administration of rhodamine 6G.
Rhodamine 6G was not mutagenic in S. typhimurium strains TA98, TA100, TA1535, or TA1537 when tested with and without exogenous metabolic activation (S9). Rhodamine 6G gave a positive response in the absence of S9 in the mouse lymphoma assay for induction of trifluorothymidine (Tft) resistance in L5178Y cells; in the presence of S9, rhodamine 6G was negative. Rhodamine 6G induced sister chromatid exchanges (SCEs) and chromosomal aberrations in cultured CHO cells in the presence, but not the absence, of S9.
Under the conditions of these 2-year feed studies, there was equivocal evidence of carcinogenic activity for male F344/N rats administered rhodamine 6G, as indicated by a marginally increased incidence of integumentary keratoacanthomas. There was equivocal evidence of carcinogenic activity for female F344/N rats administered rhodamine 6G, as indicated by a marginal increase in pheochromocytomas or malignant pheochromocytomas (combined) of the adrenal gland. There was no evidence of carcinogenic activity for male B6C3F1 mice administered 1,000 or 2,000 ppm rhodamine 6G in the diet. There was no evidence of carcinogenic activity for female B6C3F1 mice administered 500 or 1,000 ppm rhodamine 6G in the diet.
There were no significant nonneoplastic lesions attributed to rhodamine 6G administration to male or female rats or male or female mice. Male and female rats might have been able to tolerate a higher concentration of rhodamine 6G in the feed.